PRODUCT INFORMATIONDENARASE® High Salt
Recombinant Serratia marcescens endonuclease, genetically engineered for higher salt tolerance, liquid.

DENARASE High Salt is a recombinant Serratia marcescens endonuclease that is genetically engineered to enable
use at elevated salt conditions without loss of activity. Similar to the wild type, the enzyme efficiently cleaves all formsof DNA and RNA into smaller nucleotides. DENARASE High Salt shows peak performance over a wide range ofbioprocessing-relevant salt concentrations and pH conditions. This flexibility makes DENARASE High Salt the perfectsolution for the removal of process-related nucleic acids in diverse biomanufacturing processes that require thepresence of high monovalent cation levels.

1 Typical Applications
DENARASE High Salt enables optimal and cost-effective removal of nucleic acids in biomanufacturing processes thatbenefit from higher salt concentrations. The enzyme is especially suitable for the use at bioprocessing-relevant 200500 mM salt and pH 7.4 – pH 8. It is designed to improve the efficiency of various applications:•Viral Vectors for Cell & Gene Therapies•Viral Vaccines•

Viscosity reduction in Lysates•

Sample preparation in Electrophoresis and Chromatography.

2 Compliance

Version 1.0, valid as of Feb. 29, 2024

DENARASE High Salt is intended for use in research and development (R&D) applications and process developmentof biologicals such as viral vectors and vaccines. The R&D-grade DENARASE High Salt is produced under ISO 9001
standard, without the use of antibiotics, Triton X-100 and raw materials of animal origin.
The launch of the DENARASE High Salt GMP-grade for use in commercial manufacturing processes of biologicals isscheduled for Q2 2025. The manufacturing process of the DENARASE High Salt GMP-grade complies with the same
regulations and standards as our well-known wild-type GMP-grade DENARASE products. This includes manufacturing
under EU GMP conditions acc. to EU GMP regulations and distribution compliant with EXCiPACT and ANSI/NSF 363
Standard and thus also meet the requirements for Good Manufacturing and Distribution Practices (GMP/GDP) for
pharmaceutical excipients. From a technical performance perspective, DENARASE High Salt R&D- and GMP-gradeare equal.

3 Removal of DENARASE High Salt
Common purification techniques like chromatography and tangential flow filtration can be used to remove
DENARASE High Salt from process intermediates.

PRODUCT INFORMATIONDENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).

4 ELISA Kit
The amino acid sequence of the DENARASE High Salt differs from the wild-type enzyme in only a few amino acids.
For this reason, the DENARASE ELISA Kit for analysis of endonucleases from Serratia marcescens can also be used
for the detection and quantification of DENARASE High Salt. As the kit only contains a standard solution of the wildtype enzyme, it is necessary to multiply ELISA kit readings of DENARASE High Salt by a factor of 1.3 to obtainaccurate protein concentrations. Alternatively, a DENARASE High Salt standard solution can be generated.

5 Operating conditions
Like the wild-type S. marcescens endonuclease, DENARASE High Salt is a robust enzyme that shows DNA clearance
activity under various conditions. Engineered to perform under a broad range of monovalent cation concentrations(Fig. 1 & Fig. 2), the enzyme’s activity is influenced by further process parameters including temperature, pH andpresence of cofactors and inhibitory compounds. To determine optimal operating conditions, DENARASE High Salt
activity was measured under standard conditions following a protocol similar, but not identical to the c-LEcta release
test protocol for DENARASE High Salt (detailed in the DENARASE High Salt Validation Guide, Section 3.1).
Therefore, enzyme activities were normalized as indicated.

5.1 Salt concentration

Version 1.0, valid as of Feb. 29, 2024

The presence of monovalent cations dose-dependently inhibits the activity of the wild-type DENARASE enzyme.
DENARASE High Salt exhibits robust activity over a broad spectrum of sodium chloride (NaCl) concentrations (Fig.
1). The activity of the salt-adapted enzyme increases with intermediate NaCl concentrations at pH 8, representing
typical release test conditions of endonucleases. Additionally, DENARASE High Salt shows high activity across0 – 500 mM NaCl at bioprocessing-relevant pH 7.4. Based on these salt profiles we recommend using DENARASE
High Salt at salt concentrations above 200 mM and the wild-type DENARASE for monovalent cation concentrations
below 200 mM.

Fig. 1 The effect of increasing NaCl concentrations on the enzyme activity of the wild-type S. marcescens endonuclease(DENARASE) and DENARASE High Salt. The volumetric activities at 15 mM MgCl2 and pH 7.4 as well as pH 8 were measured
in U/ml. After normalization to the activities as given on the CoAs of the respective enzyme batches, enzyme activities werenormalized to DENARASE High Salt activity at 250 mM NaCl and pH 7.4 (indicated by dotted circle). The yellow and pink hatchedareas mark the recommended salt range for the use of DENARASE and DENARASE High Salt, respectively.

Compared to SAN HQ (Salt Active Nuclease High Quality, ArcticZymes), the DNA clearance activity of DENARASE
High Salt is higher over a broader salt concentration range (Fig. 2). Furthermore, DENARASE High Salt shows highDNA clearance activity at bioprocessing-relevant low pH levels (pH 7.4, Fig. 2 left). The activity can further beenhanced by increasing the presence of magnesium (see also Section 5.2.).

PRODUCT INFORMATION
2/6DENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).

Fig. 2 The effect of increasing NaCl concentrations on the enzyme activity of DENARASE High Salt and Salt Active NucleaseHigh Quality (SAN, ArcticZymes). The volumetric activities at pH 7.4 (left) and pH 8 (right) were measured in U/ml. Afternormalization to the activities as given on the CoAs of the respective enzyme batches, enzyme activities were normalized toDENARASE High Salt activity at 250 mM NaCl, 15 mM MgCl2 and pH 7.4 (indicated by dotted circle).

5.2 Magnesium
As for many other enzymes, magnesium (Mg2+) is an essential co-factor and a prerequisite for the nucleic acidclearance activity of the S. marcescens wild-type enzyme. To determine the influence of magnesium on theperformance of DENARASE High Salt, the enzyme activity was measured under standard conditions in presence of0 – 100 mM MgCl2. Requiring the presence of minimal magnesium levels for basal activity, DENARASE High Salt
shows activity at the standard condition of 5 mM MgCl2 and pH 7.4 (Fig. 3; indicated by dotted circle). Raising themagnesium levels up to 25 mM and increasing the pH value to 8.0 increases the endonuclease activity at all tested salt
concentrations (Fig. 3 & Fig. 4). However, excess of MgCl2 will reduce DENARASE High Salt activity.

Version 1.0, valid as of Feb. 29, 2024

Fig. 3 Effect of low to high MgCl2 concentrations on enzyme activity of DENARASE High Salt at 250 mM NaCl. The volumetricactivities at pH 7.4 and pH 8 were measured in U/ml and normalized to activity at 5 mM MgCl2 and pH 7.4 (indicated by dottedcircle).

Fig. 4 Effect of low to intermediate MgCl2 levels in combination with 0-500 mM NaCl on enzyme activity of DENARASE High
Salt. The volumetric activities at pH 7.4 (left) and pH 8 (right) were measured in U/ml and normalized to activity at 250 mM
NaCl, 5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

PRODUCT INFORMATION
3/6
DENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).

DENARASE High Salt activity has been measured in frequently applied buffer systems such as Tris-HCl andphosphate buffers (Fig. 5). For all tested buffers the DENARASE High Salt activity was enhanced by increasing MgCl2
concentrations, which thus can be a solution to circumvent the inhibitory effects of high phosphate concentrations.

Fig. 5 Effect of MgCl2 on DENARASE High Salt activity in Tris-HCl and potassium phosphate (KP) buffers. Volumetric activities
at 250 mM NaCl were measured in U/ml and normalized to maximal enzyme activities.

5.3 Temperature & pH
To determine the optimal reaction temperature and pH value, DENARASE High Salt activity was measured understandard conditions (250 mM NaCl, 5 mM Mg2+) at different temperatures and in several buffers at different pH values.
The optimal reaction conditions for DENARASE High Salt are 37 °C and pH 7.4 – 9.0 (see Fig. 6 & Fig. 7). Similar toother process parameters, high MgCl2 levels enhance the activity of DENARASE High Salt at all temperatures and pH
levels tested when compared to the standard magnesium levels.

Version 1.0, valid as of Feb. 29, 2024

Fig. 6 Effect of temperature on DENARASE High Salt activity.
Enzyme activity at different temperatures at pH 7.4 and 8.0
were normalized to activity at 37 °C, 5 mM MgCl2 and pH 7.4(indicated by dotted circle).

Fig. 7 Effect of pH on DENARASE High Salt activity. Enzyme
activities in Tris-HCl, Bis-Tris and potassium phosphate (KP)
buffer at different pH levels in the presence of 5 mM and 15 mM
MgCl2 were normalized to DENARASE High Salt activity at
5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

6 Stability & Storage Conditions
When stored properly (-20 °C ± 5 °C), the enzyme is stable for at least 6 months from the date of QC release.
Note: It is not recommended to store the product at -70 °C or below, since freezing the product will cause loss ofactivity. The long-term stability of DENARASE High Salt is currently under investigation.

7 Packaging Information
DENARASE High Salt is filled in non-pyrogenic, USP Class VI compliant vials. The product vials are shipped under
qualified cooled conditions. Shipping temperature may differ from the storage temperature without affecting productquality. All DENARASE High Salt products will be delivered by c-LEcta in a sealed secondary packaging with tamperevident seals.
PRODUCT INFORMATION
4/6
DENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).

8 Enzyme Characteristics
The enzyme catalyzes the hydrolysis of phosphodiesters of all forms of DNA and RNA, including single-stranded,double-stranded, linear, circular, or supercoiled forms into smaller nucleotides.
Molecular weight (calculated)

27 kDa (per monomer)

pH optimum

pH 7.4 - 9.0

Temperature optimum

37 °C

Isoelectric point (pI, calculated)

7.831

Cofactor

Mg²+1

Calculated using Clone Manager 11 Professional Edition (Sci Ed Software); reflects calculation acc. to Lehninger

9 Product Specification
Recombinant Serratia marcescens endonuclease, genetically engineered for higher salt tolerance, produced by
microbial fermentation with Bacillus sp. The production strain employed in the manufacturing of the product is a
Genetically Modified Organism (GMO) of safety level S1.
The enzyme is supplied as liquid and formulated in 20 mM Tris-HCl pH 7.4 ± 0.2, 250 mM NaCl, 5 mM MgCl2, 50 %glycerol (v/v).
Produced under ISO 9001 standard.

1

Parameter

MethodSpecification

Appearance

VisualClear, transparent solution

Activity

Photometric1> 250 U/µl

PurityProtein purity determined by SDS-PAGE and silver staining≥ 99 %

Specific Activity

Activity per protein content determined photometrically at 280 nm > 5 x 105 U/mg
with a molar extinction coefficient of 44,600 L x mol-1 x cm-1

Endotoxin level

LAL-Test
acc. to Ph. Eur. 2.6.14, Method C< 0.25 EU/kU

Total microbial count

TAMC/TYMC
acc. to Ph. Eur. 2.6.12

Aerobic bacteria: < 5 cfu/200 µl
Yeast/moulds: < 5 cfu/200 µlbnqn

Δnnn

Version 1.0, valid as of Feb. 29, 2024

Unit-Definition: One unit (U) will digest salmon sperm DNA to acidpH 8.0 at 37 °C.

PRODUCT INFORMATION
5/6
DENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).

10

Sales and Contact

Products for use in research and development
ProductArt. No

SizeActivity

DENARASE High Salt 25 kU

22002-25k

25 kU> 250 U/µL

Produced under ISO 9001 standard

DENARASE High Salt 100 kU

22002-100k

100 kU> 250 U/µL

Produced under ISO 9001 standard

DENARASE High Salt 500 kU22002-500k

500 kU> 250 U/µL

Produced under ISO 9001 standard

DENARASE High Salt 1000 kU

22002-1000k

1 MU> 250 U/µL

Produced under ISO 9001 standard

DENARASE High Salt 5000 kU

22002-5000k

5 MU> 250 U/µL

Produced under ISO 9001 standard

Contact

Version 1.0, valid as of Feb. 29, 2024

c-LEcta GmbH
Perlickstraße 504103 Leipzig
Germany
Phone:
Fax:
Mail:
Web:

+49 341 355 214-0+49 341 355 214-33
denarase@c-LEcta.com
www.denarase.com

c-LEcta GmbH Perlickstraße 5, 04103 Leipzig / c-LEcta.com

PRODUCT INFORMATION
6/6DENARASE® is a registered trademark of c-LEcta GmbH in the European Union (EU), United States of America (USA), China (CN), India(IN), South-Korea (KR) and Japan (JP).